Avijit Ray

A Novel IL-10–Independent Regulatory Role for B Cells in Suppressing Autoimmunity by Maintenance of Regulatory T Cells via GITR Ligand

B cells are important for the regulation of autoimmune responses. In experimental autoimmune encephalomyelitis (EAE), B cells are required for spontaneous recovery in acute models. Production of IL-10 by regulatory B cells has been shown to modulate the severity EAE and other autoimmune diseases. Previously, we suggested that B cells regulated the number of CD4+Foxp3+ T regulatory cells (Treg) in the CNS during EAE. Because Treg suppress autoimmune responses, we asked whether B cells control autoimmunity by maintenance of Treg numbers. B cell deficiency achieved either genetically (μMT) or by depletion with anti-CD20 resulted in a significant reduction in the number of peripheral but not thymic Treg. Adoptive transfer of WT B cells into μMT mice restored both Treg numbers and recovery from EAE. When we investigated the mechanism whereby B cells induce the proliferation of Treg and EAE recovery, we found that glucocorticoid-induced TNF ligand, but not IL-10, expression by B cells was required. Of clinical significance is the finding that anti-CD20 depletion of B cells accelerated spontaneous EAE and colitis. Our results demonstrate that B cells play a major role in immune tolerance required for the prevention of autoimmunity by maintenance of Treg via their expression of glucocorticoid-induced TNFR ligand.

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of naïve macrophages in the peritoneal cavity makes it a preferred site for the collection of naïve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Pathogenic and regulatory roles for B cells in experimental autoimmune encephalomyelitis.

A dual role of B cells in experimental autoimmune encephalomyelitis (EAE), the animal model of the human autoimmune disease multiple sclerosis (MS), has been established. In the first role, B cells contribute to the pathogenesis of EAE through the production of anti-myelin antibodies that contribute to demyelination. On the contrary, B cells have also been shown to have protective functions in that they play an essential role in the spontaneous recovery from EAE. In this review, we summarize studies conducted in a number of species demonstrating the conditions under which B cells are pathogenic in EAE. We also discuss the phenotype and anti-inflammatory mechanisms of regulatory B cells.

Cannabinoid Receptor 2 Is Critical for the Homing and Retention of Marginal Zone B Lineage Cells and for Efficient T-Independent Immune Responses

The endocannabinoid system has emerged as an important regulator of immune responses, with the cannabinoid receptor 2 (CB2) and its principle ligand 2-archidonoylglycerol playing a major role. How CB2 regulates B cell functions is not clear, even though they express the highest levels of CB2 among immune cell subsets. In this study, we show that CB2-deficient mice have a significant reduction in the absolute number of marginal zone (MZ) B cells and their immediate precursor, transitional-2 MZ precursor. The loss of MZ lineage cells in CB2−/− mice was shown to be B cell intrinsic using bone marrow chimeras and was not due to a developmental or functional defect as determined by B cell phenotype, proliferation, and Ig production. Furthermore, CB2−/− B cells were similar to wild type in their apoptosis, cell turnover, and BCR and Notch-2 signaling. We then demonstrated that CB2−/− MZ lineage B cells were less efficient at homing to the MZ and that their subsequent retention was also regulated by CB2. CB2−/− mice immunized with T-independent Ags produced significantly less Ag-specific IgM. This study demonstrates that CB2 positively regulates T-independent immune responses by controlling the localization and positioning of MZ lineage cells to the MZ.

IL-10-independent regulatory B-cell subsets and mechanisms of action

Although classically B cells are known to play important roles in immune protection via humoral immunity, recently their regulatory mechanisms have been best appreciated in the context of autoimmunity. Several studies have identified different subsets of regulatory B cells that vary not only in their phenotype but also in their mechanism of action. Although the best-studied mechanism of B-cell immune regulation is IL-10 production, other IL-10-independent mechanisms have been proposed. These include maintenance of CD4(+)Foxp3(+) regulatory T cells; production of transforming growth factor-β, IL-35, IgM or adenosine or expression of PD-L1 (programmed death 1 ligand 1) or FasL (Fas ligand). Given that B-cell-targeted therapy is being increasingly used in the clinic, a complete understanding of the mechanisms whereby B cells regulate inflammation associated with specific diseases is required for designing safe and effective immunotherapies targeting B cells.

Mechanisms of Regulatory B cell Function in Autoimmune and Inflammatory Diseases beyond IL-10

In the past two decades it has become clear that in addition to antigen presentation and antibody production B cells play prominent roles in immune regulation. While B cell-derived IL-10 has garnered much attention, B cells also effectively regulate inflammation by a variety of IL-10-independent mechanisms. B cell regulation has been studied in both autoimmune and inflammatory diseases. While collectively called regulatory B cells (Breg), no definitive phenotype has emerged for B cells with regulatory potential. This has made their study challenging and thus unique B cell regulatory mechanisms have emerged in a disease-dependent manner. Thus to harness the therapeutic potential of Breg, further studies are needed to understand how they emerge and are induced to evoke their regulatory activities.

Cannabinoid Receptor 2 (CB2) Plays a Role in the Generation of Germinal Center and Memory B Cells, but Not in the Production of Antigen-Specific IgG and IgM, in Response to T-dependent Antigens

The cannabinoid receptor 2 (CB2) has been reported to modulate B cell functions including migration, proliferation and isotype class switching. Since these processes are required for the generation of the germinal center (GC) and antigen-specific plasma and memory cells following immunization with a T-dependent antigen, CB2 has the capacity to alter the quality and magnitude of T-dependent immune responses. To address this question, we immunized WT and CB2−/− mice with the T-dependent antigen 4-hydroxy-3-nitrophenylacetyl (NP)-chicken-gamma-globulin (CGG) and measured GC B cell formation and the generation of antigen-specific B cells and serum immunoglobulin (Ig). While there was a significant reduction in the number of splenic GC B cells in CB2−/− mice early in the response there was no detectable difference in the number of NP-specific IgM and IgG1 plasma cells. There was also no difference in NP-specific IgM and class switched IgG1 in the serum. In addition, we found no defect in the homing of plasma cells to the bone marrow (BM) and affinity maturation, although memory B cell cells in the spleen were reduced in CB2−/− mice. CB2-deficient mice also generated similar levels of antigen-specific IgM and IgG in the serum as WT following immunization with sheep red blood cells (sRBC). This study demonstrates that although CB2 plays a role in promoting GC and memory B cell formation/maintenance in the spleen, it is dispensable on all immune cell types required for the generation of antigen-specific IgM and IgG in T-dependent immune responses.

Interrelatedness between dysbiosis in the gut microbiota due to immunodeficiency and disease penetrance of colitis

The composition of the microbiome in health and disease has only recently become a major research focus. Although it is clear that an imbalance or dysbiosis in the microbiota is associated with disease, its interrelatedness to disease penetrance is largely unknown. Inflammatory bowel disease (IBD) is an excellent disease in which to explore these questions because of the extensive genetic studies identifying disease susceptibility loci and the ability to easily sample the intestinal microbiota in IBD patients due to the accessibility of stool samples. In addition, mouse models of IBD have contributed to our understanding of the interrelatedness of the gut microbiota and genes associated with IBD. The power of the mouse studies is that multiple colitis models exist that can be used in combination with genetically modified mice that harbour deficiencies in IBD susceptibility genes. Collectively, these studies revealed that bacterial dysbiosis does occur in human IBD and in mouse colitis models. In addition, with an emphasis on immune genes, the mouse studies provided evidence that specific immune regulatory proteins associated with IBD influence the gut microbiota in a manner consistent with disease penetrance. In this review, we will discuss studies in both humans and mice that demonstrate the impact of immunodeficiences in interleukin‐10, interleukin‐17, nucleotide‐binding oligomerization domain (NOD) 2, NOD‐like receptor proteins 3 and 6, Toll‐like receptor or IgA have on the interrelatedness between the composition of the gut microbiota and disease penetrance of IBD and its mouse models.

An Increase in Tolerogenic Dendritic Cell and Natural Regulatory T Cell Numbers during Experimental Autoimmune Encephalomyelitis in Rras−/− Mice Results in Attenuated Disease

R-Ras is a member of the Ras superfamily of small GTPases, which are regulators of various cellular processes, including adhesion, survival, proliferation, trafficking, and cytokine production. R-Ras is expressed by immune cells and has been shown to modulate dendritic cell (DC) function in vitro and has been associated with liver autoimmunity. We used Rras-deficient mice to study the mechanism whereby R-Ras contributes to autoimmunity using experimental autoimmune encephalomyelitis (EAE), a mouse model of the CNS autoimmune disease multiple sclerosis. We found that a lack of R-Ras in peripheral immune cells resulted in attenuated EAE disease. Further investigation revealed that, during EAE, absence of R-Ras promoted the formation of MHC IIlow DC concomitant with a significant increase in proliferation of natural regulatory T cells, resulting in an increase in their cell numbers in the periphery. Our study suggests a novel role for R-Ras in promoting autoimmunity through negative regulation of natural regulatory T cell numbers by inhibiting the development of MHCIIlow DC with tolerogenic potential.